There is clear clinical evidence that genetic modulated osteoporosis results from functional defects in osteoblast cells. However, the genes underlying these genetic syndromes of osteoporosis remain largely unknown. In order to identify genes that are important for bone formation, we employed subtractive-differential screening and promoter-trap mutagenesis techniques. Surprisingly, a zinc-finger protein, cellular nucleic acid binding protein (CNBP), was identified by both approaches. We have found that the expression of CNBP gradually increases during osteoblast differentiation in vitro. In vivo, CNBP is predominantly expressed in osteoblasts, and is detected throughout the osteoblast lineage, from early osteoprogenitors to osteocytes during bone development. The CNBP homozygous mutant mice generated from promoter-trap mutagenesis die in the prenatal period and heterozygous mice survive to develop postnatal skeletal abnormalities associated with osteoporosis. Examination of mutant embryos during development indicated that impaired bone formation may be due to fewer pre-osteoblasts and osteoblasts with reduced functional capability. CNBP directly regulates osteocalcin expression and overexpression of CNBP increases osteoblast proliferation in vitro. Based on our preliminary data, we hypothesize that CNBP controls the differentiation of mesenchymal progenitor cells into mature osteoblasts and the proliferation of osteoblasts. To test this hypothesis, we propose four Specific Aims. In Aim 1, we will examine the spatial and temporal expression of CNBP, which will be correlated with bone marker gene expression during bone development and postnatal life. The expression of bone marker genes in CNBP mutants will be studied to evaluate the molecular basis of the bone defect phenotype. In Aim 2, we propose to define the functional role of CNBP in osteoblast proliferation and differentiation in vitro using forced expression and co-transfection methods. In Aim 3, to overcome the early embryonic lethality in CNBP mutants, we propose to study the role of CNBP in bone formation in vivo by conditional knockout of the CNBP gene in osteoblasts using Cre/loxP technology. The effect of the mutation on osteoblast proliferation and differentiation will be examined. In Aim 4, we will generate transgenic mice that overexpress CNBP in osteoblasts in order to examine the role of CNBP in bone formation during embryonic development and postnatal life, and in the prevention of osteoporosis using ovariectomized transgenic mice. The ultimate goal of these studies is to understand how CNBP and other bone transcription factors interact during bone development, and to apply this knowledge to develop new diagnostics and therapeutics for human osteoporosis.